The present invention, in some embodiments thereof, relates to Listeria bacteria having mutations in their multidrug resistance transporters. In some embodiments, the bacteria can be used as vaccines, adjuvants and as DNA delivery vehicles.
Strains of Listeria monocytogenes have recently been developed as intracellular delivery vehicles of heterologous proteins providing delivery of antigens to the immune system to induce an immune response to clinical conditions that do not permit injection of the disease-causing agent, such as cancer (U.S. Pat. No. 6,051,237; U.S. Pat. No. 6,565,852) and HIV (U.S. Pat. No. 5,830,702). Since L. monocytogenes is a Gram-positive, food-borne human and animal pathogen responsible for serious infections in immunocompromised individuals and pregnant women, strains of these bacteria must be attenuated in a manner that reduces toxicity to the host, while maintaining immunogenicity of the vaccine.
As a facultative intracellular bacterium, L. monocytogenes elicits both humoral and cell-mediated bacterial antigen-specific immune responses. Following entry of the Listeria into a cell of the host organism, the Listeria produces Listeria-specific proteins that enable it to escape from the phagolysosome of the engulfing host cell into the cytosol of that cell. In the cell, L. monocytogenes proliferates, expressing proteins necessary for survival, but also expressing heterologous genes operably linked to Listeria promoters. Presentation of peptides of these heterologous proteins on the surface of the engulfing cell by MHC proteins permit the development of a T cell response. During infection, L. monocytogenes triggers a robust type I interferon response, as manifested by enhanced expression and secretion of the cytokine beta interferon (IFN-β).
A previous study aimed at identifying L. monocytogenes determinants involved in IFN-β activation identified multidrug resistance (MDR) transporters as modulators of the type I interferon response in vivo (Crimmins et al., Proc. Natl. Acad. Sci. U.S.A. 105:10191-10196). Specifically, overexpression in bacteria of two closely related MDR transporters, MdrM and MdrT, was found to trigger enhanced induction of IFN-β by infected macrophages. However, only deletion of the mdrM gene resulted in reduced levels of IFN-β secreted by infected macrophages. This observation indicated that MdrM plays an active role during bacterial cytosolic growth that leads to induction of the type I interferon response.
It was recently proposed that MdrM and MdrT transporters extrude cyclic-di-adenosine monophosphate (c-di-AMP) during L. monocytogenes intracellular growth, which in turn activates infected macrophages to elicit the IFN-β response (11, 12). Indeed, c-di-AMP activates a robust type I interferon response when added exogenously, however, a physiological association between c-di-AMP and the MDR transporters was not established. Notably, several reports had indicated that c-di-AMP serves as a second messenger molecule that influences central cellular processes of bacteria e.g., genome surveillance, response to cell wall stresses and, more recently, peptidoglycan homeostasis (13-17). In bacteria c-di-AMP is synthesized by diadenylate cyclase (DAC) using ATP as a substrate and conversely, linearized to 5′pApA by a specific c-di-AMP phosphodiesterase (PDE) (15). While it was shown that the level of c-di-AMP is largely dependent on the expression levels of DAC and PDE enzymes (15, 18), the mechanism coordinating the activity of these enzymes is not known. The prevalence of DAC domains among bacteria and archaea strengthens the premise that this c-di-AMP is fundamentally involved in microbial physiology (13). L. monocytogenes genome encodes both c-di-AMP dac and pde genes (dacA: lmo2120 and pdeA: lmo0052). dacA gene was shown to be essential for growth and the one responsible for c-di-AMP production, while pdeA was shown to degrade c-di-AMP (11, 18).
Additional background art includes WO 2008/066774 and U.S. Patent Application No. 20110027319.